The goal of this proposal is to define a novel signal transduction pathway that is activated by bacterial lipopolysaccharide (LPS), but does not require signaling by Toll-like Receptor 4 (TLR4). While TLR4 is widely recognized to control transcriptional responses to LPS, its role in controlling immediate (non-transcriptional) responses is less clear. Examples of such responses include endocytosis, phagocytosis, autophagy and tyrosine phosphorylation. How LPS triggers these activities is largely unknown, but we have discovered that non-transcriptional responses control the classically-defined transcriptional responses to LPS. Understanding how microbial products activate distinct signal transduction pathways may permit the design of drugs that can target a subset of pathways therapeutically. Our proposal is founded on our recent discovery that LPS-induces the endocytosis of TLR4 by a process that does not require TLR4 signaling. Rather TLR4 is cargo for an endocytosis pathway that is activated by the LPS-binding protein CD14. While TLR4 does not direct its own endocytosis, this process is essential for TLR4 to induce TRIF-dependent signal transduction from endosomes. Our discovery of a response to LPS that does not require TLR4 signaling is important, as this receptor is thought to mediate all responses to LPS (also known as endotoxin). In this grant application, we propose to 1) identify new cytosolic regulators of CD14-dependent endocytosis, 2) identify the extracellular interactions that are needed for CD14-dependent endocytosis, and 3) identify the importance of CD14-dependent endocytosis in encounters between macrophages and pathogenic Yersinia. Collectively, this work will provide important insight into the means by which non-transcriptional responses to LPS are controlled.